Viruses have proven to be powerful tools to study fundamental cellular mechanisms. In this proposal we will focus on interactions of viruses with cellular DNA repair pathways. We have discovered that viral genomes can represent substrates for the cellular DNA repair machinery, and that viral infection can lead to activation of signaling pathways characteristic of the DNA damage response. We will use the human adeno-associated virus (AAV) as a simple model system to study interactions with cellular factors. AAV possesses a single- stranded DNA genome and a biphasic lifecycle that requires helper viruses for efficient productive infection. We have uncovered a novel cellular DNA damage response that is activated by AAV co-infection with adenovirus, involving signaling by PI3-like kinases through cellular repair proteins. We will investigate the nature of the damage response, the viral elements that trigger it, the cellular kinases involved, and its impact on virus replication. The virus lifecycle is subject to remarkable spatial and temporal regulation, with the formation of structures within the nucleus where viral replication occurs. We will develop live cell imaging approaches to visualize AAV infection and will use these to (1) detect the conversion of incoming single- stranded DNA genomes to a duplex form, (2) study the dynamics of viral replication and (3) track interactions of cellular repair factors with viral replication centers. Our studies have demonstrated that viruses provide model systems to study the cellular responses to DNA damage. The experiments proposed in this grant will address mechanistic aspects of the recognition of the AAV genome by cellular repair proteins and how this cellular response affects virus infection. We will use a combination of genetic, biochemical and cytological approaches to study these host responses to viral infection. Recombinant forms of AAV provide one of the simplest and most promising vectors for gene therapy applications. Understanding how the cell recognizes, responds and processes viral DNA, is important for any gene therapy approach that delivers genetic material in viral vectors. Therefore, in addition to providing insights into the fundamental process of recognition of damaged DNA, these studies will also have implications for genetic approaches to combat disease.